1. Field of the Invention
The present invention relates to methods of spectrophotometric determination of the presence of particular substances in a complex mixture of compounds. This invention relates to methods for determining the concentration of various components of complex biological samples including the determination of the concentration of polyoxyethylene containing compounds including polysorbate in protein containing samples as well as determining the ratio of drug to antibody ratios in antibody-drug conjugate solutions.
2. Description of the Related Art
Many compositions in the food, cosmetic and pharmaceutical industries are comprised of complex mixtures of organic molecules. For example, polysorbate is a common ingredient used in various industries including the pharmaceutical industry where it is used to stabilize drugs, including biologicals. Recent research works indicate consumption of polysorbate could cause problems in the human health, such as heart problems, infertility, blood clotting, and high blood pressure (Ema, M.; Hara, H.; Matsumoto, M.; Hirata-Koizumi, M.; Hirose, A.; Kamata, E., “Evaluation of developmental neurotoxicity of polysorbate 80 in rats”. Reproductive Toxicology 25 (1): 89-52, 2008; Oser, B L, Oser M., “Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. I. General plan and procedures; growth and food utilization”. J. Nutr. 60 (3): 367-90, November 1956; Oser, B L, Oser M., “Nutritional studies on rats of diets containing high levels of partial ester emulsifiers. II. Reproduction and lactation”. J. Nutr. 60 (4): 489-505, December 1956; Gajdová M., Jakubovsky J., Války J., “Delayed effects of neonatal exposure to Tween 80 on female reproductive organs in rats”. Food Chem. Toxicol. 31 (3): 183-90, March 1993; Williams, J, Odum J, Lewis R W, Brady A M., “The oral administration of polysorbate 80 to the immature female rat does not increase uterine weight”. Toxicol. Lett. 91 (1): 19-24, March 1997). This research has attracted the attention of both EU and USA regulatory agencies provoking discussions as to whether to monitor and regulate the content of polysorbate in the drug.
Current pharmaceutical industries standards permit polysorbate to be used in the manufacture of drugs; however, there are no standard methods to determine the exact content of polysorbate in the final drug products. The concentration of polysorbates are difficult to measure due to the fact that they are heterogeneous in nature, tend to form micelles and may bind to other components in mixtures, such as proteins, or bind to the surfaces of containers within which the polysorbates are held. Some measuring methods have been developed to determine polysorbate concentrations. (Daniel Hewitt, Taylor Zhang, Yung-Hsiang Kao, “Quantitation of polysorbate 20 in protein solutions using mixed-mode chromatography and evaporative light scattering detection”, Journal of Chromatography A, Vol 1215, Issues 1-2, 26 Dec. 2008, Pages 156-160; Lakshmy, M Nair, Norma V Stephens, Sarah Vincent, Neervalur Raghavan, Patrick J Sand, “Determination of polysorbate 80 in parenteral formulations by high-performance liquid chromatography and evaporative light scattering detection”, Journal of Chromatography A, Volume 1012, Issue 1, 12 Sep. 2003, Pages 81-86; Travis H. Tania, Jamie M. Mooreb, Thomas W. Patapoff, “Single step method for the accurate concentration determination of polysorbate 80”, Journal of Chromatography A, Volume 786, Issue 1, 24 Oct. 1997, Pages 99-106). One such method requires retreatment of the protein sample and colorimetric determination of the content of polysorbate (Alfred Weber, Andrea Engelmaier, Heinz Anderle, Hans-Peter Schwarz, “Method For The Determination Of Polysorbate80”, US Patent Publication No. 20120225487). This process involves separation, filtration, and dilution of samples, all of which is time consuming and prone to inducing errors during both sample preparation and measurement.
Antibody-drug conjugates or ADCs are a new class of highly potent biopharmaceutical drugs designed as targeted drug therapy for the treatment of people with cancer. ADCs are complex molecules composed of an antibody linked, via a stable, chemical linker with labile bonds, to a biological active cytotoxic compound. The manufacture of ADCs provides the challenge of conjugating the cytotoxic drug component to the antibody via a chemical linker in a reproducible fashion. It would be desirable to characterize the drug to antibody ratio, the amount of bound drug versus unbound drug, the amount of unbound linker and to determine the stability of the ADC both in vitro and in vivo.
Therefore, there exists a need for a method to determine the presence of particular substances in a complex mixture of compounds simply and without purification, modification or dilution. In particular, there is a need for methods which can quantitatively measure the concentration of polyoxyethylene compounds, including polysorbates, in protein samples which provide direct measurement of the samples and which do not need complicated and time consuming sample preparation. Also, there is a need to determine the concentration ratios of drug to antibody in antibody-drug conjugates (ADC).